Adenine and uridine nucleotides are released in a regulated fashion from neurons and many other cell types, and as extracellular signaling molecules convey important physiological and pathophysiological responses in essentially all tissues. The potential for therapeutic interventions at the receptors that regulate these responses is both very large and to date completely unrealized. At least six G protein-coupled P2Y receptors (P2Y-R) recognize extracellular nucleotides as their cognate agonists. The long-term objectives of this research are to define in molecular terms the interaction of P2Y-R with their agonists and downstream signaling proteins and to develop high affinity receptor- selective ligands for these receptors. The five cloned human P2Y-R (P2Y1, P2Y2, P2Y4,.. P2Y6, and P2Y11) and an avian P2Y-R (P2Ya-R) will be purified to homogeneity after baculovirus/Sf9 cell expression of tagged recombinant proteins. These purified receptors will be reconstituted in proteoliposomes with heterotrimeric Gq and their agonist selectivities determined for the first time under conditions not encumbered by problems of nucleotide metabolism and release peculiar to studies of P2Y-R in intact cells. We have discovered that ATP is a naturally occurring antagonist of the human P2Y4-R, and mutational analyses will identify the amino acid residues in the receptor binding pocket responsible for this novel activity. The G protein-selectivity of the P2Y-R will be determined by reconstitution with members from all four classes of heterotrimeric G proteins. A particular emphasis will be on the P2Ya-R, which appears to couple to both Gq and Gi, and on the human P2Y11-R, which apparently couples to both Gq and Gs. Several RGS proteins exhibit receptor-selective effects, and we recently observed that RGS9-beta5 augments receptor/G protein coupling. The receptor and G protein specificity of these RGS protein-promoted effects will be examined in proteoliposomes reconstituted with P2Y-R Identification of novel ligands for P2Y-R will continue with development of non- nucleotide antagonists, inverse agonists and radioligands for the P2Y1-R and antagonists for the P2Y4-R These approaches will result in definition of the agonist binding and G protein specificity of the P2Y-R, will provide new insight into receptor regulation by RGS proteins, and will generate much-needed new ligands that can be applied to physiological, pharmacological, and molecular studies of the P2Y-R.